阅读说明：本技术 一种增强l-精氨酸产生菌表达水平的方法 (Method for enhancing expression level of L-arginine producing strain ) 是由 杨晟 蒋宇 杨俊杰 董枫 刘映淼 于 2018-06-05 设计创作，主要内容包括：本发明公开了一种通过基因工程提高L-精氨酸产生菌生产能力的方法,包括下述步骤：敲除谷氨酸棒杆菌ATCC13032基因组中的argR基因,获得基因敲除菌株ATCC13032(ΔargR)；对基因敲除菌株的基因组中argB基因进行A26V和M31V突变,获得基因突变菌株ATCC13032(ΔargR,argBmut(A26V M31V))；对基因突变菌株的基因组中ATP依赖的CLP蛋白酶基因NCgl2585碱基序列进行E484K突变,获得基因工程菌株ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2585mut52)。本发明的方法能够将菌株的L-精氨酸生产能力提高至少15倍。(The invention discloses a method for improving the production capacity of L-arginine producing strain through genetic engineering, which comprises the following steps: knocking out argR gene in Corynebacterium glutamicum ATCC13032 genome to obtain gene knock-out strain ATCC13032 (delta argR); carrying out A26V and M31V mutation on argB gene in genome of the gene knockout strain to obtain a gene mutation strain ATCC13032 (delta argR, argBmut (A26V M31V)); E484K mutation was performed on the nucleotide sequence of the ATP-dependent CLP protease gene NCgl2585 in the genome of the gene mutant strain to obtain the genetically engineered strain ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut 52). The method of the present invention can improve the L-arginine producing ability of the strain by at least 15 times.)

1. A method for improving the productivity of L-arginine producing bacteria, comprising the following steps:

A. Knocking out argR gene in Corynebacterium glutamicum ATCC13032 genome to obtain gene knock-out strain ATCC13032 (delta argR);

B. Performing A26V and M31V mutation on argB gene in the genome of the knockout strain ATCC13032 (delta argR) in the step A to obtain a gene mutant strain ATCC13032 (delta argR, argBmut (A26V M31V));

C. E484K mutation was performed on the nucleotide sequence of the ATP-dependent CLP protease gene NCgl2585 in the genome of the genetic mutant strain ATCC13032(Δ argR, argBmut (A26V M31V)) described in step B, to obtain a genetically engineered strain ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut52) having an improved L-arginine producing ability as compared to the genetic mutant strain.

2. the method of claim 1, wherein the knockout strain ATCC13032 (aargr) in step a is prepared by:

A1. Using ATCC13032 genome as a template, and using a primer argR-aL-F with a sequence of SEQ ID NO. 1 and a primer argR-aL-R with a sequence of SEQ ID NO. 2 to carry out PCR amplification to obtain an argR-aL fragment with about 1 kb;

A2. Using ATCC13032 genome as a template, and using a primer argR-aR-F with a sequence of SEQ ID NO. 3 and a primer argR-aR-R with a sequence of SEQ ID NO. 4 for PCR amplification to obtain an argR-aR fragment with about 1 kb;

A3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

gibson ligated argR-aL, argR-aR and vector fragments as described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

A5. Carrying out PCR amplification by using primers argR-aL-F and argR-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argR;

A6. Preparing corynebacterium glutamicum ATCC13032 competent cells;

A7. Plasmid pK18mobsacB-argR was transformed into ATCC13032 competent cells;

A8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argR-aL-F and argR-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified, yielding strain ATCC13032 (. DELTA.argR).

3. The method of claim 1, wherein the genetic mutant strain of step B is prepared by:

B1. Using ATCC13032 genome as template, using primer argB-aL-F with sequence SEQ ID NO. 5 and primer argB-aL-R with sequence SEQ ID NO. 6 to carry out PCR amplification, obtaining about 1kb argB-aL fragment;

B2. Carrying out PCR amplification by using ATCC13032 genome as a template and a primer argB-aR-F with a sequence of SEQ ID NO. 7 and a primer argB-aR-R with a sequence of SEQ ID NO. 8 to obtain an argB-aR fragment with about 1 kb;

B3. The plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

Gibson ligated the argB-aL, argB-aR and vector fragments described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

B5. Carrying out PCR amplification by using primers argB-aL-F and argB-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argBmut;

B6. preparing corynebacterium glutamicum ATCC13032(Δ argR) competent cells;

B7. transformation of plasmid pK18mobsacB-argBmut into ATCC13032(Δ argR) competent cells;

B8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argB-aL-F and argB-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified to obtain strain ATCC13032(Δ argR, argBmut (A26V M31V)).

4. The method of claim 1, wherein the genetically engineered strain in step C is prepared by:

C1. PCR amplification was performed using the ATCC13032 genome as a template, and primer NCgl2585mut52-aL-F having the sequence SEQ ID NO. 9 and primer NCgl2585mut52-aL-R having the sequence SEQ ID NO. 10 to obtain about 0.5kb NCgl2585mut52-aL fragment;

C2. PCR amplification was performed using the ATCC13032 genome as a template, and primer NCgl2585mut52-aR-F having the sequence SEQ ID NO. 11 and primer NCgl2585mut52-aR-R having the sequence SEQ ID NO. 12 to obtain about 0.5kb NCgl2585mut52-aR fragment;

C3. The plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

Gibson ligated the NCgl2585mut52-aL fragment, NCgl2585mut52-aR fragment and vector fragment described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

C5. Transformants were verified by PCR amplification using primers NCgl2585mut52-aL-F and NCgl2585mut52-aR-R to give plasmid pK18mobsacB-NCgl2585mut 52;

C6. Preparing competent cells of Corynebacterium glutamicum ATCC13032(Δ argR, argBmut (A26V M31V));

C7. Plasmid pK18mobsacB-NCgl2585mut52 was transformed into ATCC13032(Δ argR, argBmut (A26V M31V)) competent cells;

C8. SacB sucrose counter-screening was performed and transformants which grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified by PCR amplification using primers NCgl2585mut52-aL-F and NCgl2585mut52-aR to obtain strain ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut 52).

5. the method of any one of claims 2 to 4, wherein the conversion in steps A7, B7, and C7 is to calcium chloride conversion or to electricity conversion.

6. A genetically engineered bacterium ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut52) constructed according to the method of claim 1.

7. The use of the genetically engineered bacterium of claim 6 for the production of L-arginine.

8. the use of claim 7, wherein the L-arginine is produced by fermentation of the genetically engineered bacteria.

9. use according to claim 7, characterized in that fermentation is carried outThe medium composition was as follows: 60g/L glucose, 5g/L corn steep liquor, 30g/L (NH)₄)₂SO₄8g/L KCl, 2g/L urea, 0.5g/L KH₂PO₄，0.5g/L K₂HPO₄，1g/L MgSO₄·7H₂O，1g/L NaCl，20mg/L FeSO₄·7H₂O，10mg/L MnSO₄·5H₂o, 20mg/L nicotinic acid, 20mg/L beta-alanine, 10mg/L VB1, 0.2mg/L biotin, 30g/L CaCO₃the pH value was adjusted to 7.7 with KOH.

10. The use according to claim 7, wherein the seed medium consists of: 3g/L NaCl, 5g/L yeast extract, 7g/L beef extract, 10g/L peptone and 10g/L glucose.

Technical Field

The invention belongs to the field of genetic engineering, relates to a method for enhancing the expression level of L-arginine producing bacteria, and particularly relates to a method for improving the production capacity of the L-arginine producing bacteria through genetic engineering.

Background

L-arginine (L-argnine, L-Arg for short) is one of semi-essential basic amino acids needed in human body, is an important intermediate metabolite of organism urea circulation as a basic amino acid containing guanidyl, has various unique physiological and pharmacological effects, has good curative effects on treating physiological functions, cardiovascular diseases, stimulating immune system, maintaining nutrition balance of infants, promoting detoxification of human body and the like, is called as an important carrier for transporting and storing amino acid in the body by experts, and is extremely important in intramuscular metabolism. It is an essential amino acid for the synthesis of cytoplasmic and nuclear proteins; participate in creatine synthesis as the only ammonia source; as an important intermediate of the urea cycle, the urea cycle plays a role in removing excessive ammonia in the liver and prevents poisoning caused by excessive accumulation of ammonia; it also has effects in regulating immunity, inhibiting tumor growth, and promoting wound tissue healing. Arginine is a direct precursor of nitric oxide, urea, ornithine and myostatin, an important element in the synthesis of myostatin, and is used as a synthesis of polyamines, citrulline and glutamine. Therefore, the L-arginine has important and wide application in the fields of medicine, food and chemical industry. For example, in clinic, besides being one of the main components of compound amino acid infusion, L-arginine and its salts are widely used for treating various hepatic coma patients who are forbidden to use sodium glutamate and viral hepatic glutamic pyruvic transaminase abnormal patients, and have obvious curative effect on viral hepatitis. It can be used for treating intestinal ulcer, thrombosis, and neurasthenia. In addition, the L-arginine is an important component of a sports nutritional beverage formula, is also an important feed additive, and is widely applied to high-end breeding industry. Statistically, the worldwide demand for L-arginine is currently over 15000 tons, and the demand increases at a rate of 12% -15% per year.

There are two methods for producing L-arginine: the first is protein hydrolysis extraction method, and the second is microorganism fermentation method. The hydrolysis method has the problems of time-consuming operation, low yield and output, high cost and the like, and has serious pollution which is not suitable for large-scale production. The fermentation method for producing the L-arginine has relatively simple process and is environment-friendly, so the method has great development potential and becomes an important trend of the domestic and foreign amino acid industry. International famous amino acid companies such as Japanese monosodium glutamate, Synergestin and Degaosha in Germany mainly use biological fermentation and genetic engineering techniques for L-arginine production. However, the acid production level of L-arginine produced by domestic microbial fermentation is generally low, the cost is high, and the production level and the yield can not meet the domestic requirements far, so the research for improving the L-arginine fermentation level has important significance.

Most of L-arginine fermentation strains are mainly Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Corynebacterium crenatum (Corynebacterium crenatum), Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis) and the like, but currently, microorganism strains for producing L-arginine are mainly Corynebacterium glutamicum and Corynebacterium crenatum. The genetic engineering technology has an important promoting effect on the breeding of the arginine high-producing strain, the construction of the L-arginine high-producing strain by utilizing the genetic engineering is a high-efficiency and rational breeding means, but the construction and screening of the high-producing recombinant strain suitable for industrial scale production is always an urgent need.

Disclosure of Invention

In order to overcome the defect of low fermentation level of the existing L-arginine producing strain, the invention utilizes the genetic engineering technology to modify corynebacterium glutamicum, and can greatly improve the L-arginine production capacity of the strain by enhancing genes related to the production of L-arginine and weakening branch metabolic pathways.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving the productivity of L-arginine producing bacteria, comprising the following steps:

A. Knocking out argR gene in Corynebacterium glutamicum ATCC13032 genome to obtain gene knock-out strain ATCC13032 (delta argR);

B. Performing A26V and M31V mutation on argB gene in the genome of the knockout strain ATCC13032 (delta argR) in the step A to obtain a gene mutant strain ATCC13032 (delta argR, argBmut (A26V M31V));

C. E484K mutation was performed on the base sequence of the ATP-dependent CLP protease gene (NCgl2585) in the genome of the gene mutant strain ATCC13032(Δ argR, argBmut (A26V M31V)) described in step B to obtain a genetically engineered strain ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut52) having an improved L-arginine producing ability as compared to the gene mutant strain.

In one embodiment, the knockout strain ATCC13032(Δ argR) described in step a above is prepared by the following method:

A1. Using ATCC13032 genome as a template, and using a primer argR-aL-F with a sequence of SEQ ID NO. 1 and a primer argR-aL-R with a sequence of SEQ ID NO. 2 to carry out PCR amplification to obtain an argR-aL fragment with about 1 kb;

A2. Using ATCC13032 genome as a template, and using a primer argR-aR-F with a sequence of SEQ ID NO. 3 and a primer argR-aR-R with a sequence of SEQ ID NO. 4 for PCR amplification to obtain an argR-aR fragment with about 1 kb;

A3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

gibson ligated argR-aL, argR-aR and vector fragments as described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

A5. carrying out PCR amplification by using primers argR-aL-F and argR-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argR;

A6. Preparing corynebacterium glutamicum ATCC13032 competent cells;

A7. Plasmid pK18mobsacB-argR was transformed into ATCC13032 competent cells;

A8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argR-aL-F and argR-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified, yielding strain ATCC13032 (. DELTA.argR).

In one embodiment, the genetic mutant strain described in step B above is prepared by:

B1. using ATCC13032 genome as template, using primer argB-aL-F with sequence SEQ ID NO. 5 and primer argB-aL-R with sequence SEQ ID NO. 6 to carry out PCR amplification, obtaining about 1kb argB-aL fragment;

B2. carrying out PCR amplification by using ATCC13032 genome as a template and a primer argB-aR-F with a sequence of SEQ ID NO. 7 and a primer argB-aR-R with a sequence of SEQ ID NO. 8 to obtain an argB-aR fragment with about 1 kb;

B3. the plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

Gibson ligated the argB-aL, argB-aR and vector fragments described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

B5. Carrying out PCR amplification by using primers argB-aL-F and argB-aR-R to verify a transformant, and obtaining a plasmid pK18 mobsacB-argBmut;

B6. preparing corynebacterium glutamicum ATCC13032(Δ argR) competent cells;

B7. Transformation of plasmid pK18mobsacB-argBmut into ATCC13032(Δ argR) competent cells;

B8. SacB sucrose counter-screening was performed, PCR amplification was performed using primers argB-aL-F and argB-aR-R, and transformants that grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified to obtain strain ATCC13032(Δ argR, argBmut (A26V M31V)).

In one embodiment, the genetically engineered strain described in step C above is prepared by:

C1. PCR amplification was performed using the ATCC13032 genome as a template, and primer NCgl2585mut52-aL-F having the sequence SEQ ID NO. 9 and primer NCgl2585mut52-aL-R having the sequence SEQ ID NO. 10 to obtain about 0.5kb NCgl2585mut52-aL fragment;

C2. PCR amplification was performed using the ATCC13032 genome as a template, and primer NCgl2585mut52-aR-F having the sequence SEQ ID NO. 11 and primer NCgl2585mut52-aR-R having the sequence SEQ ID NO. 12 to obtain about 0.5kb NCgl2585mut52-aR fragment;

C3. The plasmid pK18mobsacB with GenBank accession number FJ437239.1 is digested with HindIII and EcoRI, and the 5.7kb vector fragment is obtained by gel recovery;

Gibson ligated the NCgl2585mut52-aL fragment, NCgl2585mut52-aR fragment and vector fragment described above, transformed DH 5. alpha. competent cells, plated on kanamycin LB plates, and cultured overnight;

C5. Transformants were verified by PCR amplification using primers NCgl2585mut52-aL-F and NCgl2585mut52-aR-R to give plasmid pK18mobsacB-NCgl2585mut 52;

C6. Preparing competent cells of Corynebacterium glutamicum ATCC13032(Δ argR, argBmut (A26V M31V));

C7. Plasmid pK18mobsacB-NCgl2585mut52 was transformed into ATCC13032(Δ argR, argBmut (A26V M31V)) competent cells;

C8. SacB sucrose counter-screening was performed and transformants which grew on BHIS plates but could not grow on kanamycin-containing BHIS plates were verified by PCR amplification using primers NCgl2585mut52-aL-F and NCgl2585mut52-aR to obtain strain ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut 52).

The conversion into calcium chloride or into electrical conversion, preferably into electrical conversion, as described in the above steps A7, B7 and C7.

According to a second aspect of the present invention, there is provided a genetically engineered bacterium ATCC13032(Δ argR, argBmut (A26V M31V), NCgl2585mut52) constructed according to the above-described method.

according to a third aspect of the present invention, there is provided the use of the above genetically engineered bacterium in the production of L-arginine.

The genetically engineered bacterium can be directly used as a fermentation strain to produce the L-arginine through fermentation, and can also be used as an original strain to be further improved so as to screen out a new production strain with further improved L-arginine production capacity.

When L-arginine is produced by fermentation of the above genetically engineered bacterium, the medium used in the fermentation may be any medium suitable for growth and fermentation of Corynebacterium glutamicum.

According to a preferred embodiment of the invention, the fermentation medium consists of: 60g/L glucose, 5g/L corn steep liquor, 30g/L (NH)₄)₂SO₄8g/L KCl, 2g/L urea, 0.5g/L KH₂PO₄，0.5g/L K₂HPO₄，1g/L MgSO₄·7H₂O，1g/L NaCl，20mg/L FeSO₄·7H₂O，10mg/L MnSO₄·5H₂O, 20mg/L nicotinic acid, 20mg/L beta-alanine, 10mg/L VB1, 0.2mg/L biotin, 30g/L CaCO₃The pH value was adjusted to 7.7 with KOH.

in a preferred embodiment, the fermentation of the L-arginine producing bacteria comprises a seed culture stage and a bacterial fermentation stage. The two stages use seed culture medium and fermentation culture medium, which may be the same as or different from the seed culture medium.

preferably, when the fermentation medium is not the same as the seed medium, the seed medium consists of: 3g/L NaCl, 5g/L yeast extract, 7g/L beef extract, 10g/L peptone and 10g/L glucose.

the method can improve the L-arginine production capacity of the original strain Corynebacterium glutamicum ATCC13032 for producing L-arginine by 15 times, and has popularization and promotion values.

drawings

FIG. 1 is a schematic representation of plasmid pK18mobsacB, which is presented by Liu Shuangjiang, institute of microbiology, academy of sciences of China. GenBank: FJ 437239.1. For more detailed information seehttps://www.ncbi.nlm.nih.gov/nuccore/ 215434894。

FIG. 2 is a schematic diagram of a recombinant plasmid pK18mobsacB-argR constructed according to the present invention.

FIG. 3 is a schematic diagram of recombinant plasmid pK18mobsacB-argBmut constructed according to the present invention.

FIG. 4 is a schematic diagram of recombinant plasmid pK18mobsacB-NCgl2585mut52 constructed according to the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

In this context, the terms "Corynebacterium glutamicum ATCC 13032", "strain ATCC 13032" and "ATCC 13032" mean the same meaning, and all refer to the original strain ATCC13032 which is the subject of genetic modification, which is an L-arginine producing strain purchased from the Shanghai institute of Industrial microbiology.

Herein, the terms "gene knock-out bacterium" and "ATCC 13032(Δ argR)" mean the same meaning, and both refer to a strain in which the argR gene in the ATCC13032 genome is knocked out.

Similarly, the terms "genetically mutant strain", "ATCC 13032(Δ argrgbbut (a26V M31V)", "ATCC 13032(Δ argR, argBmut (a26V M31V)" and "CIBTS 1512" mean the same meaning, and refer to strains in which the argBmut gene is mutated to the argBmut (a26V M31V) gene in the genome of gene knockdown sterilized ATCC13032(Δ argR).

The terms "genetically engineered bacterium (strain)", "ATCC 13032(Δ arggBmut (A26V M31V) NCgl2585mut 52)", "ATCC 13032(Δ argR, argBmut (A26V M31V), NCgl2585mut 52)", "CIBTS 1512(NCgl2585mut 52)" and "CIBTS 3010" mean strains in which the nucleotide sequence SEQ ID NO:13 of the ATP-dependent CLP protease gene (NCgl2585mut (A26V M31V)) is further mutated to NCgl2585mut52 gene in the genome of the genetically mutated bacterium ATCC13032(Δ argR, argBmut (A26 3585M 31V)), so that the glutamic acid (E) at the amino acid position of CLP protease 484 is substituted with lysine (K).

The gene argR is an arginine operon regulating gene which is ubiquitous in bacteria and has different functions in different bacteria.

N-acetylglutamate kinase (N-acetylglutamate kinase) has a catalytic function for the first step of the arginine synthesis pathway from glutamate to acetylglutamate by C.glutamicum without feedback inhibition by the end product L-arginine. The inventors have found that L-arginine synthesizing ability of Corynebacterium glutamicum ATCC13032 can be effectively improved by mutating N-acetylglutamate kinase to change alanine (A) at position 26 to valine (V) and methionine (M) at position 31 to valine (V). Herein, the mutant gene of the N-acetylglutamate kinase gene argB is abbreviated as argBmut or argBmut (A26V M31V).

It was unexpectedly found that a change in the base sequence of the ATP-dependent CLP protease gene (NCgl2585), SEQ ID NO:13, can cause a change in the arginine synthesizing ability. The mutation of the L-arginine-containing amino acid sequence into SEQ ID NO. 14 can improve the L-arginine yield of the corynebacterium glutamicum to a certain extent.

Experiments show that the L-arginine production capacity of the gene mutant strain CIBTS1512 is improved by at least 13 times compared with that of the original strain ATCC 13032; the L-arginine production capacity of the genetic engineering strain CIBTS3010 is improved by 14.6 percent compared with that of CIBTS1512, namely the production capacity of CIBTS3010 is improved by at least 15 times compared with that of the original strain ATCC13032, and the correctness of the genetic engineering design concept of the invention is verified.